presumably nitrates by acid-catalyzed formation of the nitronium ion:
H+
+ KO-NO2 + A c ~ O+ Noif + 2HOAc
(5)
T o control the nitration to avoid production of yellow nitrated pyrene compounds (IC), triethyl phosphate solvent was used to prepare 1.6M and 0.2M nitric acid reagents. Turbak (19) has found that 1 : l complexes of triethyl phosphate and sulfur trioxide will not attack aromatic rings but will react with other functional groups. The 1.6M reagent (1.5:1 triethyl phosphatenitric acid) did attack carbazole but not pyrene as shown in Table XI. It would appear as though the phosphoryl oxygen of triethyl phosphate controls the reactivity of nitric acid because reagents containing 0.5:1 or 1: 1 triethyl phosphate-nitric acid did react rapidly with pyrene. The 0.2144 reagent was mild enough to reduce the interference of 2naphthylamine, as shown in Table XI, without producing the excessive colored oxidation products encountered with the more concentrated reagents. To eliminate slow side reactions of nitric acid, excess methanol was added to react with the nitric acid and thus quench the reaction ( 7 ) :
NO2+
+ MeOH
+
MeONOz
+ Hf
(6)
This was critical in the oxidation of phenyl sulfide with the 2.3-44 reagent to avoid appreciable nitration of pyrene. ACKNOWLEDGMENT
The authors thank Norman Radke for investigating the fluorescence of the TNF-pyrene .complex, Jean Jessup for some experimental measurements, hlilagros Santiago for preliminary work on the nitric acid-acetic anhydride reagent, and Howard Simmons of E. I. du Pont de Nemours & Co. for furnishing a sample of tetracyanothiophene. LITERATURE CITED
(1) Briegleb, G., “Elektronen-Donator-
Acceptor Kornplexe,” p. 176, SpringerVerlag, Berlin, 1961. (2) Cundiff, R. H., Markunas, P. C., ANAL.CHEM.35, 1323 (1963). (3) Drago, R. S., Rose, N. J., J . Am. Chem. Soc. 81, 6141 (1959). (4) Gordon, H. T., Huraux, M. J., ANAL. CHEM.31, 302 (1959). (5) Klemrn, L. H., Sprague, J. W., J . Org. Chem. 19, 1464 (1954). (6) Lepley, A. R., J . Am. Chem. SOC.84, 3577 (1962).
(7) Malins, D. C., Wekell, J. C., Houle, C. R.,ANAL.CHEM.36, 658 (1964). (8) Melby, L. R., Harder, R. J., Hertler, W. R., Mahler, W., Benson, R. E., Mochel, W. E., J . Am. Chem. SOC.84, 3374 (1962). (9) Merrifield, R. E., Phillips, W. D., Ibid., 80, 2778 (1958). (10) Orchin, At., Reggel, L., Woolfolk, E. 0.. Ibid.. 69. 1225 (1947). (11) Orchin, 61., Woolfolk, E.‘ O., Ibzd., 68, 1727 (1946). (12) Ozolins, M., Schenk, G. H., ANAL. CHEM.33, 1035 (1961). (13) Ray, K. E., Francis, W. C., J . Org. Chem. 8. 52 (19431. (14) Sawicki, E., Stanley, T. W., ChemistAnalyst 49, 77 (1960). (15) Schenk, G. H., Fritz, J. S., ANAL. CHEM.32, 987 (1960). (16) Schenk, G. H., Santiago, M., Wines, P., Zbid., 35, 167 (1963). (17) Siggia, S., Stahl, C. R., Ibid., 35, 1740 i19631. (18) Tamres,’M., J . Phus. Chem. 65,654 ( 1961). (19) Turbak, A. F., Division of Polymer Chemistry, ACS, Preprints 2, S o . 1, 140 (1961); through C.A. 57: 153319. RECEIVEDfor review August 10, 1964. Accepted December 17, 1964. Work supported by Public Health Research Grant GM-07760 from the National Institutes of Health, Public Health Service. Presented in part at the 15th Pittsbureh Conference on Analvtical Chemistry and Applied Spectroscopy, March 1964.
Isolation and Identification of Alcohols in Cold-Pressed Valencia Orange Oil by Liquid-Liquid Extraction and Gas Chromatography G.
L.
K. HUNTER and M. G. MOSHONAS
Fruit and Vegefable Products laboratory, Winter Haven, Flu.
b A procedure for the qualitative analysis of alcohols in cold-pressed Valencia orange oil is presented. The method utilizes the partitioning of the oil between carbon tetrachloride and propylene glycol. Water is added to the propylene glycol layer and the essential oil alcohols are extracted with ether. The alcohols are further purified by column chromatography, separated by gas chromatography, and identified by infrared and mass spectrometry. Nineteen alcohols were isolated from Valencia cold-pressed orange oil and identified as n-octanol; n-decanol; linalool; citronellol; a-terpineol; nnonanol; trans-carveol; geraniol; nerol; heptanol; undecanol; dodecanol; elemol; cis- and frans-2,8-pcis-carveol; 1 -pmenthadiene-1-01; methene-9-01; 1,8-p-menthadiene-9-01; and 8-p-methene-1,2-diol. The first nine alcohols have been reported to be in orange oil or essence by others. 378
ANALYTICAL CHEMISTRY
The remaining 10 have not been reported to be constituents of orange oil, and the last four alcohols of these 10 have not been reported in natural products.
V
INVESTIQATORS attempted to determine the alcohols present in citrus essential oils or essence directly from mixtures either by column chromatography (7, 8), by column chromatography followed by gas chromatography on total terpenoids (2), or by subtractive analyses of the whole oil in conjunction with gas chromatography (15). Recently, Attaway et al. (1) described a procedure in which alcohols were isolated from orange essence oil through removal of the carbonyls by Girard-T reagent ( I $ ) followed by solvent gradient elution through columns of either activated alumina or silicic acid. Frilette, Mower, and Ruben (4) have subsequently shown that the conARIOUS
ditions under which the Girard-T procedure is performed are sufficient to promote dehydration of some tertiary alcohols. The present paper describes a procedure for isolation of the alcohols from cold-pressed orange essential oil using the nonaqueous extraction procedure of Suffis and Dean (12) with some modifications. Yonalcoholic materials which resulted from incomplete partitioning were removed from the alcohol fraction by column chromatography. The alcohols were subsequently separated by gas chromatography and identified spectrometrically. The partitioning of citrus oils between carbon tetrachloride and propylene glycol is particularly effective because the major portion of the oil is nonalcoholic and expeditiously removed by the carbon tetrachloride. The alcohols are isolated from the oil by preferential solubility in propylene glycol, eliminating the danger of artifact formation.
The extractive procedure is relatively fast and only a relatively small amount of oil is needed to obtain a sufficient quantity of material for analysis by gas chromatography, This procedure makes monitoring of alcohols possible when studying the changes which take place in flavor under various conditions of processing and storage.
c
EXPERIMENTAL
0
Apparatus.
Column chromatoggraphy was accomplished in a 6/g- X 18-inch column containing basic alumina. The hydrocarbons and carbonyls were separated from the crude alcoholic extract, which had been placed on t h e column, by elut'ion with a 50/50 solution of n-hexane and ethyl ether. The alcohols remaining on the column were desorbed with absolute ethanol. The alcohols were separated by gas chromatography in an F & 1LI Model 500 gas chromatograph containing a l/l-inch 0.d. by 20-foot aluminum tube packed with 17y0 Carbowax-3011 on acid-washed Chromosorb-G. The oven temperature was programmed from 150' to 220' C. at 1.1' C. per minute with a helium flow rate of 54 ml. per minute. Fractions were collected directly onto micro salt plates ( 5 ) for infrared analysis and mass spectra were obtained on the same material following infrared analysis. Larger quantities of material were collected when necessary in small vials immersed in a vapor bath of liquid nitrogen. Mass spectra were obtained using a Bendix Model 12-100 whose source was operated a t 70 e.v. and recorded on an EA1 X-Y Variplotter. Infrared spectra were obtained using a Perkin-Elmer Model 137 Infracord spectrometer and, when necessary, critical absorption assignments were determined on a Beckman IR-7 spectrometer. Procedure. The alcohols were enriched by an adaption of the carbon tetrachloride-propylene glycol partition method of Suffis and Dean (12). One liter each of orange oil and carbon tetrachloride and 500 ml. of propylene glycol were vigorously shaken in a 4liter separatory funnel for 20 minutes. The two layers were separated and the propylene glycol portion was centrifuged to remove insoluble materials and residual carbon tetrachloride. One liter of a sat'urated aqueous solution of sodium chloride was added to the glycol and the resulting solution was extracted with four 150-ml. portions of ethyl et.her. The combined ether extracts were washed with three 500-ml. portions of water and dried over anhydrous sodium sulfate. T h e ether was removed under reduced pressure to yield 11.0 grams of residue. The alcohol fraction was further purified by column chromatography. ,4 3.5-gram portion of the residue was placed on a basic alumina column and eluted with a 100-ml. solution of nhexane and ethyl ether to remove the nonalcoholic materials. The alcohols were stripped from the column with 100
Figure 1 .
chromatogram of 35-pl. sample of alcohol fraction
ml. of absolute ethanol to yield 1.5 grams of residue upon removal of the solvent under reduced pressure. The alcohols were separated by gas chromatography and the materials represented by the peaks were collected and analyzed. When two alcohols are represented by a single peak in the chromatograms, such as linalool-octanol and trans-carveol-geraniol, the material represented by each side of the peak was collected to yield each component sufficiently pure for identification by infrared and mass spectrometric analyses. RESULTS A N D DISCUSSION
Combination of the liquid-liquid extraction procedure and column chromatography of the whole essential oil simplified the gas chromatographic separation of the alcohols by removal of other functional group components. The chromatogram shown in Figure 1 was obtained from a 35-pl. sample of the alcoholic fraction and the names of 19 compounds are indicated thereon. The alcohols n-octanol, n-nonanol, n-decanol, citronellol, linalool, and aterpineol, which have previously been identified as constituents of orange oil (1, 8) are shown to be present by comparison of infrared spectra. Geraniol, nerol, and truns-carveol, which have been tentatively identified to be in orange oil by other workers (1, 2, 8),are now confirmed as a result of separation and purification by gas chromatography and comparison of their infrared and mass spectrometric properties with published data (14). The remaining 10 alcohols (n-hep-
tanol; n-undecanol; n-dodecanol; cisand trans-2,8-p-menthadiene-l-ol; elemol; cis-carveol; 1-p-menthene-9-01; 1$-p-menthadiene-g-ol; and 8-p-menthene-1,2-diol) are reported here to be constituents of Florida cold-pressed Valencia orange oil for the first time. The last four alcohols have not been found before in natural products. The three aliphatic alcohols, n-heptanol, undecanol, and dodecanol, were identified by comparison of their respective infrared and mass spectra and gas chromatographic retention data with that obtained on materials of known structure. The infrared spectra of trans- and cis-2,8-p-menthadiene-l-ol show a terminal double bond absorption a t 890 and 1640 em.-', and a disubstituted double bond absorption at 735 cm.-l for the trans isomer and 745 em.-' for the cis isomer. Their molecular weights were determined to be 152 by mass spectrometry, indicating a monocyclic terpene alcohol containing two double bonds. The hydroxyl group was established to be in the one position by catalytic reduction with platinum oxide and hydrogen a t 70 p.s.i. to yield l-p-menthol. The tentative structures were confirmed by direct comparison with infrared spectra kindly provided by h'aves and Grampoloff and described by them (9). The structure of elemol, a sesquiterpene alcohol, was indicated by the nature of its mass spectrometric cracking pattern. The mass spectrum showed the loss of water by the appearance of the molecular ion a t m/e 222 and a peak a t 204. The cracking patVOL. 37, N O . 3, MARCH 1965
* 379
tern of the remaining spectrum was characteristic of elemene (6). In addition, the infrared spectrum of elemol was identical to that published by Pliva et al. (11 ) . The identity of cis-carveol was indicated by the similarity of its infrared spectrum to that of trans-carveol. In addition, the mass spectrum of the uncharacterized alcohol is all but identical to that of trans-carveol. The infrared and mass spectra and gas chroniatographic retention time of cis-carveol, obtained by the reduction of carvone with lithium aluminum hydride, were identical to the material in question. The possibility that cis-carveol resulted from the isomerization of trans-carveol during gas chromatographic analysis was ruled out because each gave only a single peak during the purification procedure. The structure of 1-p-menthene-9-01 was proved ~y infrared and mass spectrometry. The infrared spectrum of 1-p-menthene-9-01 showed an absorption a t 802 cm.-l, indicating a trisubstituted double bond. I t s molecular weight, determined by mass spectrometry, is 154 and, upon reduction with PtO, and hydrogen a t 70 p.s.i., increased to 156, indicating a monocyclic terpene alcohol with one double bond. The infrared spectrum of the completely reduced alcohol was identical to that of 9-menthol. The infrared spectrum of the unknown terpene alcohol was identical to the spectrum obtained on
an authentic sample of l-p-menthene-
LITERATURE CITED
9-01 kindly provided by Brown (3). The identity of lJ8-p-menthadiene-901 was established by comparison with 1-p-menthene-9-01 following reduction of the terminal double bond of the former, infrared absorption a t 900 and 1648 cm.-’, with P t black and hydrogen a t 70 p.s.i. The molecular weight of 152 for the diene, 154 for the monoolefin after reduction of the terminal double bond, and 156 for the totally reduced derivative, as a result of reduction with PtOp and hydrogen a t 70 p.s.i., were determined by mass spectrometry. The infrared spectrum of the totally reduced derivative is identical to that of 9-p-menthol. The identity of the material represented by the last peak in Figure 1 was shown to be 8-p-menthene-l,2-diol. Information on the structure was indicated by the appearance of three key m/e values in its mass spectrum; 170 represents the molecular ion; 152, the loss of a mol cule of water; and 134, the loss of a second molecule of water. Infrared analyses showed the presence of a terminal double bond by absorption at 1640 and 918 cm.-l The data indicated the compound to be a terpene diol with a terminal double bond. The mass and infrared spectra and gas chromatographic retention time of 8-pmenthene-1,2-diol, obtained by the hydration of limonene oxide (IO), were identical to that obtained from the orange oil.
(1) Attaway, J. A,, Wolford, R. W,, Alberding, G. E., Edwards, G. J., J . Agr. Food Chem. 12, 118 (1964). (2) Bernhard, R. A., J . Food Sci. 26, 40
(1961).
(3) Brown, H. C., Sweifel, G., J . Am. Chem. SOC.83. 1241 (19611.
(4) Frilette, V. J., Mower, E. B., Ruben, M. K., J . Catalysis 3, 25 (1964). (5) Hunter, G. L. K., Appl. Spectry. 18, 159 (1964). (6) Hunter, G. L. K., Brogden, W. B., ANAL.CHEW36, 1122 (1964). (7) Ikeda, R. &Spitler, I,, E. M., J. Agr. Food Chem. 12, 114 (1964). (8) Kirchner, J. G., &filler, J. Rf., Zbid., 5 , 283 (1957). (9) Naves, Y. R., Grampoloff, A. V., Bull. SOC.Chim. France 1960, p. 37. (10) Newhall, W. F., J . Org. Chem. 23, 1274 11958). (11) Plivai-J:, Horak, M., Herout, V., Sorm, F., “Die Terpene,” Vol. 1, Sesquiterpenes, Akademie, Verlog, Berlin, 1960. (12) Suffis, R., Dean, D. E., ANAL.CHEW 34, 480 (1962). (13) Teitelbaum, C. L., J . Org. Chem. 23, 646 (1958). (14) Von Sydow, Erik, Acta Chem. S c a d . 17, 2504 (1963). (15) Wolford, R. W., Alberding, G. E., Attaway, J. A , , J . Agr. Food Chem. 10, 297 (1962). RECEIVED for review December 1, 1964. Accepted December 31, 1964. Fruit and Vegetable Products Laboratory, Winter Haven, Fla., is alaboratory of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. Mention of brand names is for identification purposes only and does not constitute endorsement by the U. S. Department of Agriculture.
Gas Chromatographic Determination of Free Formic and Acetic Acids in Cigarette Smoke E. T. OAKLEY, LUDWIG WEISSBECKER, and F. E. RESNIK Philip Morris Research Center, Richmond, Va. Free formic and acetic acids and their salts in cigarette smoke may b e quantitatively determined in the presence of formates and acetates after adsorbing the acids on a strong base anion exchange resin in the fluoride form. The compounds are eluted and methylated in a single step with an anhydrous methanol-hydrochloric acid solution. The methyl esters are subsequently determined by gas chromatography. This method i s applicable to problems requiring the analysis of free formic and acetic acids in the presence of their esters.
D
of free formic and acetic acids in complex mixtures containing formates and acetates has long been a problem. Most methods reported for the analysis of formic and
380
acetic acids require alkali which hydrolyzes the esters of these acids. Formates and acetates are easily hydrolyzed even when passed through a strong base anion exchange resin in the OH- form, The method described here is for the separation of free formic and acetic acids in cigarette smoke extracts, without hydrolyzing the esters, by adsorption onto a strong base anion exchange resin in the fluoride form, followed by simultaneous methylation and elution from the resin. The resulting methyl esters are separated and quantitatively determined by gas chromatography.
ETERMINATION
ANALYTICAL CHEMISTRY
EXPERIMENTAL
Reagents. A strong base anion exchange resin, Rexyn 202 (CI-SO4) from Fisher Scientific Co. is used.
Anhydrous methanol-hydrochloric acid solution. Anhydrous hydrochloric acid (The Matheson Co., East Rutherford, N. J.) is bubbled into anhydrous methanol (J. T. Baker Chemical Co., Phillipsburg, N. J.) so that the solution contains 7 to 10% HCI. The concentration of hydrochloric acid is checked by titration of an aliquot with 0.1.V NaOH to the methyl red end point. Potassium fluoride solution. Reagent grade potassium fluoride from Fisher Scientific Co. is used to prepare a 10% aqueous solution. One milliliter of a solution containing 2.5 mg. per milliliter of formic acid (Fisher Scientific Co.) and 5.0 mg. per milliliter of acetic acid (Baker and Adamson Chemical Co.) is used for recovery studies. The exact concentrations of the acids are determined by titration with 0.IN KaOH to the methyl red end point.
